Although estrogen (E) administration prevents bone loss and osteoporosis in postmenopausal women, the mechanism of its action at the cellular and molecular level remains unclear. The osteoblasts (OB) have been shown by many laboratories to contain estrogen receptors (ER) and to be target cells for E, but the exact actions of E on bone cells have been obscured by varying concentrations of ER and E responses between different OB cell lines. The roles of the ER-beta species and progesterone (P) in OB cells further complicate the issue. In order to discern the effects that the varying concentrations of ER and the ER-alpha and beta species have on the biological actions on E on OB cells, our laboratories have generated and characterized over a dozen conditionally immortalized, mature, human OB cell lines (hFOB) which display normal OB-like properties. These cells have been stably transfected to express varying levels pf ER-alpha (hFOB/ER-alpha), ER-beta (hFOB/ER-beta), or both species, and several express endogenous PR. We have also developed and characterized six lines of immortalized precursor cells from human bone marrow stroma 9hMS), which can differentiate into either the OB ro adipocyte phenotype. These hFOB/ER and hMS cell lines will be used to answer the following questions: 1) are mature OB or their precursors potential target cells for E, and if so, what concentrations of ER and which ER species (alpha or beta) are required for the regulation of early and late genes, including cytokines and bone matrix proteins, as well as OB functions, such as cell proliferation, matrix production, and mineralization; 2) are both PRA and PRB species expressed in the OB or their precursor cells, does the PRA:PRB ratio change during OB differentiation and differ among the ER-alpha or ER-beta containing cells, and which genes/processes do these PR species regulate; 3) do E and P regulate OB precursor cell (hMS) differentiation, including gene expression; and finally 4) are the potent new OB-derived soluble, neutralizing receptor, osteoprotegrin, and its ligand (OPGL), regulated by E in the hFOB/ER cells and do they in turn mediate the E action on osteoclast cell differentiation and activity.